MUTAGENESIS STUDIES OF INTERLEUKIN-8 - IDENTIFICATION OF A 2ND EPITOPE INVOLVED IN RECEPTOR-BINDING

Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a vari ety of cells and which elicits proinflammatory responses from the neut rophil. As a prelude to drug design, we have investigated the interact ions between IL-8 and its receptor by preparing a number of single sit e mutants of IL-8 and determining their activity in receptor-binding a nd functional assays, In order to define the binding surface as precis ely as possible, we have used chemical shifts obtained from nuclear ma gnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation, In addition to a previously recognized sequence, Glu(4)- Leu(5)-Arg(6) in the N-terminal peptide, we have identified a second e pitope comprising a contiguous group of non-sequential, solvent-expose d, hydrophobic residues, Phe(17), Phe(21), Ile(22), and Leu(43). These two receptor-binding regions are separated by over 20 Angstrom in the IL.8 structure and are important both for receptor binding and functi on. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biol ogical activity.